The ultimate goal of this proposal is to dissect the relationship between gene expression and mRNA secondary structure. Evidence has been accumulating rapidly over the past few years that the impact of mRNA structure on gene expression is profound. For example, the relative stability of mRNA secondary structures near the ribosomal binding site - the Shine-Dalgarno (SD) sequence - and resultant modulation in translation initiation appear to dominate gene expression in bacteria. More specifically, bacterial riboswitches that involve refolding of non-coding RNA secondary and tertiary structures upstream of the SD sequence in response to binding of a small metabolite control the expression of up to 4% of all genes in some bacteria. These observations have profound impact on: (i) the optimal heterologous over-expression of recombinant proteins in bacteria; (ii) the design of effective antibiotics that interfere with bacterial mRNA (re)folding in a way that traps the RNA in a deleterious conformation; and (iii) the prospects of a synthetic biology that rewires or builds bacterial gene expression circuitry for applications in, for example, bioremediation. While a plethora of crystal structures and in-solution probing data of the ligand-binding aptamer domains of riboswitches have recently emerged, few mechanistic studies have focused on the functionally critical communication of the aptamer with the downstream expression platform within the native mRNA under controlled conditions that include elements of the gene expression machinery. We here propose to refocus our long-standing expertise (established over the past three funding cycles) in dissecting the mechanism of small non-coding RNAs onto a pair of mRNAs hosting small 7- aminomethyl-7-deazaguanine (preQ1) sensing riboswitches that control their translation initiation. Our tool set ranges from novel single molecule fluorescence assays to in vitro translation and solution footprinting. By targeting both a crystallized thermophilic preQ1 riboswitch and a homology-modeled preQ1 riboswitch from the mesophilic pathogen Bacillus anthracis, we will be able to compare the dependence of their folding pathways on their differential mRNA sequences and the environmental conditions employed (Specific Aim 1). Hypothesizing that binding of the 30S ribosomal subunit to the mRNA is vulnerable to competition by ligand-mediated riboswitch folding only at a very early stage of translation initiation, we will probe the mechanistic parameters determining the outcome of this functional competition for both riboswitches (Specific Aim 2). Finally, we will probe the functional roles of competition of the 30S ribosomal subunit with other cellular factors for binding of the mRNA, as well as the impact of molecular crowding (Specific Aim 3). Several key features of these preQ1 riboswitches are exemplary for functional non-coding RNAs, including their metal-dependent hierarchical folding of secondary before tertiary structure, their use of an intricate hydrogen-bonding network to effect docking of distal domains, and the coexistence of multiple functionally relevant conformational isomers. We thus anticipate that our results will significantly deepen our understanding of the biological function of structured RNAs in general.